An adverse tumor-protective effect of IDO1 inhibition

Summary By restoring tryptophan, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors aim to reactivate anti-tumor T cells. However, a phase III trial assessing their clinical benefit failed, prompting us to revisit the role of IDO1 in tumor cells under T cell attack. We show here that IDO1 inhibition leads to an adverse protection of melanoma cells to T cell-derived interferon-gamma (IFNγ). RNA sequencing and ribosome profiling shows that IFNγ shuts down general protein translation, which is reversed by IDO1 inhibition. Impaired translation is accompanied by an amino acid deprivation-dependent stress response driving activating transcription factor-4 (ATF4)high/microphtalmia-associated transcription factor (MITF)low transcriptomic signatures, also in patient melanomas. Single-cell sequencing analysis reveals that MITF downregulation upon immune checkpoint blockade treatment predicts improved patient outcome. Conversely, MITF restoration in cultured melanoma cells causes T cell resistance. These results highlight the critical role of tryptophan and MITF in the melanoma response to T cell-derived IFNγ and uncover an unexpected negative consequence of IDO1 inhibition.


In brief
Whereas IDO1 inhibitors were developed to reinvigorate T cells by restoring tryptophan, they lack clinical benefit. Kenski et al. discover an adverse effect of IDO1 inhibition: protecting melanoma cells from the effects of T cell-derived IFNg. MITF plays a key role in this response in vitro and in patients.

INTRODUCTION
Despite the profound improvement in melanoma outcome upon immune checkpoint blockade (ICB), therapy resistance limits clinical benefit for many patients. 1 This creates a need not only for uncovering additional targets for immunotherapy, but also for a better understanding of the mechanisms of action of their corresponding inhibitors. An example is indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme induced by interferon-gamma (IFNg) 2 and responsible for catalyzing the conversion of essential amino acid tryptophan to kynurenine. 3 Pre-clinical evidence suggested IDO1 as a key mechanism of acquired immune tolerance, by affecting several immune cells within the tumor microenviron-ment. 4,5 IDO1 expression and consequently low tryptophan levels increases intratumoral T regulatory cell (Treg) and myeloid-derived suppressor cell (MDSC) infiltration while decreasing dendritic cell (DC) antigen uptake, mediating IFNg-induced differentiation of monocytes into M2 macrophages and impairing cytotoxic T cell function. 5,6 Therefore, IDO1 inhibitors were developed and evaluated in combination with pembrolizumab (anti-programmed cell death-1/PD-1 antibody) in ECHO-301/KEYNOTE-252, a large phase 3 trial. Whereas the aforementioned pre-clinical evidence supported the use of IDO1 inhibition in the context of immunotherapy in solid tumors, 7,8 co-treatment of pembrolizumab with epacadostat failed to improve progression-free survival compared with pembrolizumab alone 9 . These results led several pharmaceutical companies to scale down or terminate their IDO1 inhibitor studies. 10 While several explanations were given for these disappointing results, including the potential low dose of epacadostat and selection bias, the reasons for the failure are still under debate. 10 Interestingly, whereas T cells are sensitive to tryptophan (TRP) deprivation in the tumor microenvironment (TME), 6,11 studies from the 80s and 90s suggested that, in fact, tumor cells, too, require TRP for viability. [12][13][14] These seemingly contradictory observations prompted us to study the functional interactions between IDO1, epacadostat, tumor cells, and T cells in more mechanistic detail.

TRP restoration by IDO1 inhibition protects tumor cells from T cell-mediated killing
To study under defined conditions the effect of IDO1 inhibition on tumor cells that are under T cell attack, we made use of a co-culture system that we previously established. 15 Briefly, we introduced the melanoma antigen recognized by T cell (MART-1)-specific T cell receptor (TCR; 1D3 clone) recognizing the human leukocyte antigen (HLA)-A2-restricted MART-1 peptide (amino acid [aa] [26][27][28][29][30][31][32][33][34][35] into CD8 + T lymphocytes isolated from blood of healthy donors. 16 To ensure specific and equal recognition by MART-1 T cells and exclude potential confounding effects of differences in IFNg signaling, antigen presentation machinery, and major histocompatibility complex (MHC) and antigen expression levels, we ectopically expressed through lentiviral transduction HLA-A*02:01 and MART-1 in a panel of human melanoma cell lines, including patient-derived melanoma xenograft (PDX) cell lines. 17 After confirming that the killing was TCRspecific (Figure S1A), we observed that exposure of these cell lines to the matched T cells led to common upregulation of IDO1 protein (albeit to varying degrees; Figure 1A). In parallel, we determined the relative susceptibility of this cell line panel to T cell-mediated killing. We observed a range of sensitivities, with some cell lines being highly sensitive, some showing an intermediate sensitivity, and others being relatively resistant ( Figure 1B).
Because IDO1 activity leads to degradation of TRP in the TME, we measured the level of TRP in the culture medium after T cell treatment of melanoma cultures at a 1:10 T cell:tumor ratio, where we observed the biggest range of tumor sensitivities. The sensitivity of cell lines to T cell killing correlated significantly with the degree of TRP drop: the most sensitive cell lines had the highest relative decline in TRP levels after T cell challenge (Figure 1C), illustrating that melanoma cells can differentially suffer from low levels of TRP, while there may also be a contribution of the expression levels of IDO1 ( Figure 1A).
We next asked whether the IDO1-induced TRP loss in fact contributed to the anti-tumor effect of IFNg. As expected, treatment with IDO1 inhibitor epacadostat led to complete restoration of TRP levels ( Figure 1D). More importantly, this was accompanied by a full rescue of the toxic effects of IFNg (Figures 1E and S1B). IDO1 inhibition also rescued from T cell-induced killing ( Figure S1C).
This result led us to investigate whether it could be recapitulated in vivo in a humanized adoptive cell transfer (ACT) setting. We used immunocompromised NOD/SCID IL2Rg null (NSG)

Figure 1. Tryptophan restoration by IDO1 inhibition protects tumor cells to T cell-mediated killing
Melanoma cells were co-cultured with MART-1 T cells (or no T cells as a control) at 1:5 and 1:10 T cell:tumor cell ratios for 24 h. (A) After co-culture, cells were harvested and immunoblotted for IDO1 (short exposure and long exposure), all in parallel; HSP90 served as loading control. (B) The same melanoma cell line panel was exposed to MART-1 T cells at indicated T cell:tumor cell ratios or no T cells as a control and stained with crystal violet after 6 days, and the percentage of surviving melanoma cells was quantified. Color coding indicates sensitivity to T cells: blue, relatively T cell-sensitive; orange, intermediate phenotype; pink, relatively resistant. Color coding was done arbitrarily for better visualization. The grouping of cell lines was not used for further analysis, and cell lines were always analyzed individually. (C) Spearman correlation between relative (to control) tryptophan drop upon T cell exposure and percentage of surviving cells after T cell challenge, both at a 1:10 (T cell:tumor cell) ratio from the experiment shown in (B). Tryptophan (TRP) levels were measured from supernatant of melanoma and MART-1 T cell co-cultures by a fluorometric assay after 24 h of the experiment shown in (B). (D) TRP concentrations from supernatants in (E) were measured by a fluorometric assay after 72 h of treatment. Statistical significance shown for the IFNg-only group was tested comparing the IFNg group against its control, whereas in the epacadostat-treated groups, it was compared with the corresponding IFNg dose. The y axis shows normalized TRP levels compared with control. (E) Cell lines were treated with IFNg (2.5, 5, or 10 ng/mL) and/or epacadostat (2 mM), fixed and stained with crystal violet after 6 days. Quantification in Figure S1B. (F) NSG mice received A375-MelanA cells subcutaneously into the flank, and after 3 days, 5 million MART-1-specific or control (untransduced) CD8 + T cells were injected intravenously, and the mice were treated daily with epacadostat (100 mg/kg) orally. n = 6 mice for T cell control group and n = 10 mice for MART-1 T celltreated group. Tumor sizes at endpoint are shown.  Figures 1F and S1D). However, recapitulating our in vitro data, we did not observe better tumor control upon epacadostat treatment in vivo, but instead a moderate, yet significant, increase in tumor expansion ( Figures 1F and S1D). In an immunocompetent B16-OVA melanoma model, while we did not observe tumor acceleration upon IDO1 inhibition (likely because of simultaneous tumor cell and immune cell protection), there was again no improved tumor control ( Figure S1E), in line with a previous study. 18 We confirmed by antibody depletion that CD8 T cells contributed to tumor control in this model ( Figure S1F).
To determine whether this effect of epacadostat was mediated by TRP, we replenished this amino acid in the culture medium in vitro. TRP restoration, too, was able to revert the anti-tumor effect of either T cells or IFNg treatment ( Figures 1G, 1H, and S1G). This result is in concordance with the rescue in TRP levels caused by epacadostat treatment ( Figure 1D) and indicate that the protection observed after IDO1 inhibition by this compound is indeed due to a specific TRP restoration. IFNg was the major contributor of the T cell effect in this setting, because its blockade by a specific antibody significantly both rescued the decline in TRP and protected tumor cells ( Figures 1I, 1J, and S1H). From these observations, we conclude that whereas IDO1 inhibitors were developed to reinvigorate immune cells in a TRP-deprived milieu, another consequence of TRP replenishment is that tumor cells are protected against T cell elimination or, in other words, an on-target adverse effect of IDO1 inhibition.
IFNg-induced TRP depletion triggers general translation stalling associated with an activating transcription factor-4 (ATF4) stress response To validate these findings in a more clinically relevant setting, we treated a panel of PDX melanoma cell lines with IFNg and again observed intrinsic differences in their susceptibility to it (Figure 2A). To better understand the transcriptional reprogramming induced by this cytokine, we performed RNA sequencing of this PDX panel as a function of IFNg treatment. Gene set enrichment analysis (GSEA) of their differentially expressed genes revealed enrichment of four distinct clusters of semantically related ontology terms: immune cell activity, protein regulation, response to cytokine, and cell death ( Figure 2B).
These results prompted us to investigate whether these IFNg-induced transcriptomic changes were related to each other and to IFNg sensitivity. In line with our previous findings (Figure 1), we detected a strong correlation between gene sets related to TRP metabolic processes, IFNg response-related pathways, cell death, and sensitivity ( Figure 2C). Furthermore, pathway changes associated with a decrease in translation were also linked to higher expression of genes involved in TRP catabolism in this dataset ( Figure 2C). Further exploring the differential response of sensitive and resistant cell lines to IFNg, we observed that signatures for amino acid deprivation, TRP catabolism/metabolism, stress response, and death-related pathways were enriched in IFNg-sensitive PDX cell lines (Figure 2D). This was also seen for a signature comprising translation initiation genes that are downregulated.
After demonstrating in PDX cell lines that IFNg induces not only substantial transcriptional changes but also affects protein translation, we set out to confirm the latter observation in our original cell line panel ( Figure 1) using ribosome profiling. We selected an IFNg-responsive human melanoma cell line (D10), which showed a steep decline in TRP levels upon T cell and IFNg co-culture ( Figures 1C and 2E). As a control, we used an IFNg-resistant cell line (888Mel), which failed to degrade TRP when encountering either IFNg or T cells ( Figures 1C and 2E). We recently demonstrated by differential ribosome codon reading (diricore) analysis that 48 h of IFNg treatment leads to stalling at the TRP codon. 21 We show here that this is preceded by a reduced signal in the initiator ATG (first methionine) at position 12 at 20 h, when there are no significant changes yet in the  Figures 2F and 2G). A slower translation initiation rate with elongation proceeding at a normal pace ( Figure 2G) causes an imbalance resulting in a significant change in ribosome occupancy at the ATG initiator, which indicates a strong general inhibition of translation, 20 here shown for IFNg-treated D10 cells. This occurred when TRP levels were low due to IFNg treatment ( Figure 2E). Strikingly, co-treatment with epacadostat fully prevented protein translation shutdown ( Figures 2F  and 2G). Diminished translation also correlated with IFNg responsiveness, because control IFNg-resistant 888Mel cells failed to show this ( Figures 2F and 2G).
A previous study has shown that cells under amino acid starvation stress can impair translation while selectively increasing translation of ATF4, 22 a key factor integrating protein translation and stress signaling, including amino acid deprivation. 23 To determine whether this was the case, GSEA was performed on the most IFNg-sensitive vs. most IFNg-resistant PDX cell lines. As expected, we observed an enrichment of IFNg and ATF4 signatures in the sensitive cell lines ( Figure 2H). Likely, the mechanism by which the sensing of uncharged tRNA-TRP in D10 cells treated with IFNg occurs is via the general control nonderepressible-2 (GCN2), leading to eukaryotic initiation factor 2 (eIF2alpha) phosphorylation and ATF4 expression. 24,25 In cells not depleted for TRP (in 888Mel cells or D10 cells treated with epacadostat), there are no uncharged tRNA TRPs and thus no increase in ribosome accumulation at the start codons and, therefore, no ATF4-induced stress response.
MITF contributes to melanoma T cell sensitivity ATF4 induction upon nutrient-deprivation stress has been associated with a reduction in the expression levels of microphtalmiaassociated transcription factor (MITF), whereby the latter is transcriptionally suppressed by the first. 26 Furthermore, MITF is a critical survival factor for melanoma 27,28 and modulates the response to targeted tumor inhibitors. [29][30][31] These data, as well as the function of MITF in phenotypic plasticity and therapeutic resistance, 32 prompted us to investigate if it has a role in a tumor/T cell context. We observed distinct melanoma cell responses to T cell challenge: whereas several cell lines showed downregulation of MITF, others failed to diminish its expression ( Figure 3A). The differential regulation of MITF was independent of activation of the early IFNg signaling cascade because all examined cell lines expressed IFNg receptors 1 and 2, as well as their downstream targets Janus kinase 1 and 2 (JAK1 and JAK2) and signal transducer and activator of transcription 1 (STAT1) (Figure S2A), while STAT1 was phosphorylated ( Figure 3A).
This diversity in MITF response and its involvement in melanoma survival led us to explore any causal relationship with the observed differential susceptibility to T cell-induced cytotoxicity. We confirmed that the sensitivity of the tumor cells to T cells was strongly associated with their ability to downregulate MITF ( Figure 3B). This correlation was also seen in the IFNg-treated PDX cell line panel when analyzed by RNA sequencing ( Figure 3C).
The inverse correlation between MITF expression and T cell susceptibility raised the possibility that the protection to IFNg by IDO1 inhibition is due to the absence of an MITF downregulation. To investigate this, we co-treated melanoma cells with IFNg and epacadostat. We observed that IDO1 inhibition prevented an ATF4-driven stress response and consequently MITF downregulation, even in the presence of active IFNg signaling, as judged by STAT1 phosphorylation ( Figure 3D). This result indicates that IFNg-induced MITF regulation is a consequence of the modulation of endogenous TRP levels.
To determine whether the downregulation of MITF upon ribosome stalling was IFNg dependent, we engineered IFNg receptor 1 (IFNgR1) knockout clones and exposed them to T cells. After co-culture, MITF downregulation did not occur when IFNg signaling was lacking ( Figure 3E). These phenomena together resulted in a profound resistance to T cell cytotoxicity ( Figure 3F).
To examine whether MITF plays a causal role, we prevented its downregulation in IFNg-sensitive cells by introducing a cassette driving moderate expression of MITF. Because we used a heterologous promoter, MITF levels remained stable upon treatment with IFNg ( Figures 3G and 3I). The IFNg signaling cascade was activated in both control and MITF-expressing cells, as  (Figures 3G and 3I). Importantly, the enforced inability of (patient-derived) melanoma cells to downregulate MITF diminished their sensitivity to IFNg (Figures 3H and 3J). These data demonstrate that the ability of melanoma cells to downregulate MITF is essential for their intrinsic susceptibility to the anti-tumor effect of IFNg.
On-treatment MITF downregulation predicts clinical outcome of ICB To determine whether these results bear clinical relevance, we analyzed MITF expression as well as its target genes in The Cancer Genome Atlas (TCGA) melanoma cohort. This revealed that MITF expression, together with that of its transcriptional targets, is inversely correlated with IFNg itself, an IFNg signature, and the IFNg-inducible gene IDO1. This inverse correlation was also seen for ATF4, amino acid deprivation, and TRP catabolism genetic signatures ( Figure 4A), in accordance with our previous observations in cell lines ( Figure 2C). Since T cells can be reinvigorated and triggered to produce IFNg by ICB, we subsequently analyzed two independent gene expression datasets of anti-PD-1-treated patients for whom both preand on-treatment samples were available. 34,35 In line with our in vitro data, those patient tumors that responded to immunotherapy downregulated MITF target genes on treatment in both cohorts, whereas the non-responding tumors did not ( Figure 4B). Similar results were observed for anti-cytotoxic T lymphocyteassociated protein 4 (CTLA-4)-treated patients 38 ( Figure S3A). We confirmed these findings in patient cohorts analyzed at the single-cell level. 36 We observed that melanoma cells in patients responding to treatment had significantly lower levels of MITF and MITF target genes than prior to treatment or non-responders ( Figures 4C and 4D). Additional mining of the Riaz 35 and Guide 34 datasets revealed that melanomas with a high IFNg signature 37 and the strongest change in MITF are most likely to respond to anti-PD-1 ( Figures 4E and 4G, left panels). Responding tumors also showed significantly higher expression levels of genes related to TRP catabolism than the other samples ( Figures 4E and 4G, right panels). Furthermore, stress response, IFNg signaling, and TRP metabolism/catabolism apoptosis-related gene sets were enriched on treatment in responders to anti-PD-1 treatment (Figures 4F and 4H). Together, these results demonstrate that the intrinsic sensitivity of melanoma cells to IFNg, T cells, and PD-1 blockade correlates with their ability to show a dynamic MITF response.

DISCUSSION
We uncovered an unexpected, and undesired, on-target effect of IDO1 inhibition: whereas IDO1 inhibitors were developed to protect cytotoxic T cells and other immune cells against the deleterious effects of TRP depletion in the TME, we demonstrate here that IDO1 inhibition (or TRP replenishment) also leads to protection of melanoma cells from T cell elimination in vitro and in vivo. Given that IFNg can exert a strong bystander effect, influencing not just antigen-positive cells, 39 the protection by epacadostat may extend to remote tumor cells. While we do not wish to claim that this is a major cause for the failure of the ECHO-301 IDO1 trial, 9,40 our results do shed light on a critical aspect of IDO1 inhibition that was not previously appreciated. This may be an opportunity to guide the design of any future immunotherapy application of IDO1 inhibitors.
Our data suggest that targeting MITF could be a promising approach in combination with immunotherapy, since its downregulation in melanoma cells under T cell attack contributes to their propensity to be eliminated. MITF is an important survival factor for melanoma, and changes in its expression levels can have major consequences in several contexts. 28,32 However, to study this in vitro is challenging, as was noted by us and many other groups: melanocytes and melanoma cells do not tolerate strong modulations of the expression levels of MITF whether by depletion or overexpression. 32, [41][42][43][44] This notwithstanding, our data are consistent with the notion that MITF is a key regulator of differential cell states when cells experience various types of stress. 32, 45,46 Additionally, chronic exposure of melanoma cells to T cells can lead to de-differentiation and resistance. 47 Taken together, our data suggest that patients with immunotherapy-refractory melanoma could benefit from an acute therapy-induced decrease of MITF levels to increase their susceptibility to cytotoxic T cells.
The clinical benefit of epacadostat was investigated in the context of anti-PD-1. 9 This is in keeping with the increasing awareness that for most advanced cancers, therapy resistance Article ll OPEN ACCESS limits the benefit of single-agent therapies. Therefore, thousands of clinical trials are currently testing combination treatments, often with anti-PD-1 (or variations thereof). As we recently argued, however, with the increasing numbers of (immuno)therapeutics developed, the possibilities for combination treatments dramatically outnumber the patients available to enroll in clinical trials. 48 Rational design, informed by fundamental biological and mechanistic insight, will be required to solve this clinical problem. The current study provides an example of how a better mechanistic understanding may contribute to this in that it not only uncovers an on-target adverse effect of IDO1 inhibition but also raises the possibility that pharmacologic MITF intervention might be explored to improve immunotherapy outcome of patients with melanoma.

Limitations of the study
This study has a number of limitations that should be considered. This includes the technical challenge of manipulating MITF in a stable and reliable manner to provide genetic confirmation of some of our findings. Similarly, while our data suggest that targeting MITF might be beneficial in combination with IDO1 inhibition, the lack of a specific MITF inhibitor precludes pharmacologic testing. Furthermore, our findings may be particularly relevant to IFNg-rich tumors and MITF-expressing melanomas. More complex immunocompetent models will be required for formal testing of our hypothesis in vivo that the positive effect that IDO1 inhibition may have on CD8 T cells is counteracted by the tumor protection described here. Lastly, datasets from patient melanomas with acquired ICB resistance and IDO1 inhibition are scarce, hindering such clinical corroboration.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank all members of the Peeper and Schumacher laboratories for helpful advice, and especially Susan van Hal for technical help during revision. We are also grateful to Riccardo Mezzadra and Raquel Gomez-Eerland for contributing to the setup of the melanoma:T cell co

DECLARATION OF INTERESTS
D.S.P. is co-founder, shareholder, and advisor of Immagene, which is unrelated to this study.  Figure 1F and Extended data Figure 1D), A375 cells (which are endogenously HLA-A2-positive) ectopically expressing Melan-A were used. The in vivo experiment from Extended Data Figure 1E and 1F, B16-F10 melanoma cells expressing the protein ovalbumin (B16-OVA) were used.

Animal studies
Animal work procedures were approved by the animal experimental committee of the NKI and performed in accordance with ethical and procedural guidelines established by the NKI and Dutch legislation. 1 million tumor cells per mouse were mixed in 50uL PBS+50uL Matrigel (Corning) prior to injection. Vehicle and Epacadostat (MedChem) were administered after diluting in DMSO + Cremophor EL (2:1) with saline addition just before use (to a final concentration of 2:1:8). Adoptive cell transfer was performed in male NSG (JAX, bred at NKI) mice from 8-12 weeks. Briefly, human A375 melanoma cells ectopically expressing MelanA were injected subcutaneously into the flank. Five million transduced MART-1-specific T cells or control T cells (CD8 + T cells that were not transduced with MART-1-specific TCR) were injected at d3 via tail vein followed by intraperitoneal injection of 100.000U of IL-2 (Proleukin) at d3-5 to support T cells. From d3 treatment with either vehicle or Epacadostat (100 mg/kg) was performed daily by oral gavage until tumors reached 1000 mm 3 . In experiment shown in Figures 1F and S1D, mice were sacrificed when tumor size reached 1000 mm 3 . For the experiments depicted in Extended Data Figure 1E, male C57BL/6JRj (8-12 weeks-old, from Janvier) mice were injected with B16-OVA cells (300.000 cells, also 1:1 PBS in Matrigel) and treated with epacadostat as described above, starting from day one. In Extended Data Figure 1F, each mouse received 100ug of CD8-depleting antibody or isotype control (BE0061, BE0090, from BioXCell), once a week prior to tumor cell injection until endpoint. CD8 + T cell depletion was confirmed by flow cytometry. After that, the experiment was conducted as described for Extended Data Figure 1E. All animals are housed in disposable cages in the laboratory animal center of the NKI, minimizing the risk of cross-infection, improving ergonomics and obviating the need for a robotics infrastructure for cage-washing. The mice were kept under specific pathogen free (SPF) conditions. Tumor growth rates were analyzed by measuring tumor length (L) and width (W), and calculating volume through the use of the formula 1/2 3 length (mm) 3 width (mm). The experiments were finished for individual mice either when the total tumor volume exceeded 1000 or 1400 mm3, when the tumor presented ulceration, in case of serious clinical illness, or when tumor growth assessment had been completed.

Lentivirus production
For virus production, HEK293T cells were transfected with the plasmid of interest and the helper plasmids (pMDLglpRRE, pHCMV-G and pRSVrev) with polyethylenimine. The day after, cells were refreshed and 24h later, culture supernatant was filtered and snap frozen for later infection. After overnight lentiviral infection, puromycin at 1 mg/mL (Sigma) or hygromycin at 5 mg/mL (Life technology) was added for selection.

Cytotoxicity assays
Cytotoxic assays were performed in 12-well plate format. Tumor cells (40-